Flutamide Metabolism in Four Different Species in Vitro and Identification of Flutamide Metabolites in Human Patient Urine by High Performance Liquid Chromatography/Tandem Mass Spectrometry

Abstract

ABSTRACT:A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono-and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

Bioactivation of Flutamide Metabolites by Human Liver Microsomes

“…P450-mediated desaturation is well documented in the literature (Ortiz de Montellano, 1995). This bioactivation pathway is consistent with the recent finding that a mercapturic acid conjugate of a hydroxylated flutamide was detected (Tevell et al, 2006). Although the authors proposed a different structure with a hydroxyl group on the tertiary carbon and a mercapturic acid attached to the primary carbon of the isopropyl group, several common MS fragment ions such as m/z 323, 289, 259, and 205 were present in the spectra of both G1 and the mercapturic acid conjugate, suggesting that the mercapturic acid conjugate was a processed product of G1 in vivo via hydrolysis and acetylation.…”

“…The major involvement of CYP1A2 in the formation of G1 could be due to the predominant role of CYP1A2 in catalyzing the formation of G1 precursor M5. A hydroxyflutamide mercapturic acid conjugate was detected in the urine of prostate cancer patients treated with flutamide (Tevell et al, 2006). Based on the similarity of MS data, this mercapturic acid conjugate is most likely derived from G1.…”

“…Recently, a GSH conjugate of hydroxylated flutamide has been detected in human liver microsomal and hepatocyte incubations (Soglia et al, 2006;Kostrubsky et al, 2007). Similarly, Tevell and coworkers (Tevell et al, 2006) have detected a mercapturic acid conjugate of hydroxylated flutamide in the urine of prostate cancer patients. In a previous study of the bioactivation of flutamide in human liver microsomes, a novel N-S glutathionyl adduct was detected and fully characterized by MS and NMR, which is formed by the direct bioactivation of the parent drug (Kang et al, 2007).…”

“…Thus, it is highly probable that metabolites of flutamide can also be bioactivated and contribute to flutamide-induced hepatotoxicity. Major metabolites of flutamide identified in vitro or in vivo so far are shown in Scheme 1 Tevell et al, 2006). In addition to 2-hydroxyflutamide, other oxidative metabolites are monohydroxylated flutamide M5, dihydroxylated flutamide M7, and trihydroxylated flutamide M8.…”

Antiandrogen flutamide affects folliculogenesis during fetal development in pigs

“…Flutamide, a non-steroid antiandrogen, binds to and blocks androgen receptors (ARs) (Tevell et al 2006). As we showed previously flutamide applied in utero on critical days of gestation alters expression of androgendependent genes during postnatal life (Durlej et al 2011a(Durlej et al , 2011b.…”